Anti-Rab5/RAB5A Antibody (monoclonal, 3E9)

• Catalog Number: orb527052
• Category: Antibodies
• Description: Anti-Rab5/RAB5A Antibody (monoclonal, 3E9). Tested in Flow Cytometry, IF, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.
• Species/Host: Mouse
• Clonality: Monoclonal
• Clone Number: Clone: 3E9
• Tested applications: FC, ICC, IF, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Mouse IgG2b
• Immunogen: A synthetic peptide corresponding to a sequence at the C-terminus of human Rab5, different from the related mouse and rat sequences by three amino acids.
• Antibody Type: Primary Antibody
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 24 kDa
• UniProt ID: P20339
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: Ras-related protein Rab-5A; RAB5A; RAB5
• Note: For research use only
• Application notes: Western blot, 0.1-0.5μg/ml Immunocytochemistry/Immunofluorescence, 2μg/ml Flow Cytometry (Fixed), 1-3μg/1x106 cells. Add 0.2ml of distilled water will yield a concentration of 500μg/ml
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of A431 cells using anti-Rab5/RAB5A antibody. Overlay histogram showing A431 cells (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Rab5/RAB5A Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

IF analysis of RAB5A using anti-RAB5A antibody. RAB5A was detected in immunocytochemical section of MCF7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL mouse anti-RAB5A Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of Rab5/RAB5A using anti-Rab5/RAB5A antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates, Lane 2: human placenta tissue lysates, Lane 3: human A549 whole cell lysates. Lane 4: human A375 whole cell lysates, Lane 5: human SW620 whole cell lysates, Lane 6: human U20S whole cell lysates, Lane 7: human HEK293 whole cell lysates, Lane 8: human U87 whole cell lysates, Lane 9: rat brain tissue lysates, Lane 10: mouse brain tissue lysates, Lane 11: mouse Neuro-2a whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-Rab5/RAB5A antigen affinity purified monoclonal antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for Rab5/RAB5A at approximately 24KD. The expected band size for Rab5/RAB5A is at 24KD.